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YK011 Mouse C-Peptide I EIA

Contents

T

. Introduction

U

. Characteristics

V

. Composition

W

. Method

X

. Notes

Y

. Performance Characteristics

Z

. Stability and Storage

References

YK011 Mouse C – Peptide I EIA

T

. Introduction

This enzyme immunoassay (EIA)kit is a stable and convenient assay system for mouse

C-peptide I in its plasma, serum and urine.

The processing of proinsulin, which occurs within the B cell, yields insulin and C-peptide

and insulin and C-peptide are secreted in equimolar quantities into blood circulation.

Therefore, the measurement of C-peptide in blood reflects the concentration of insulin and

also provides valuable information to evaluate the pancreatic B cell function.

The EIA kit is prepared by using synthetic mouse C-Peptide I as standard antigen and

biotinylated mouse C-Peptide I as labeled antigen. The kit contains specific polyclonal

antibody recognized to the amino acid sequence of mouse C-Peptide.

Now, we have still tried to develop mouse C-peptide II EIA kit and mouse C-peptide I+II

kit in our laboratory.

U

. Characteristics

This ELISA kit is used for quantitative determination of mouse C-Peptide in its plasma,

serum & urine samples. It has a lot of advantage to perform the assay, such as good

quantification, no influence with other body fluid factors or physiogical active substances

and needlessness of sample pretreatment. Mouse C-Peptide I standard is highly purified

synthetic product ( purity: higher than 98% ) and biotinylated peptide is stable because N

–biotinylglycylglycyl Mouse C-Peptide I is used for it.

Specificity

The EIA kit shows each cross reactivity of 6.3% to mouse C-Peptide II, 6.5% to mouse

insulin, 156.4% to rat C-Peptide I, 114.6% to rat C-Peptide II and 7.4% to rat insulin and

shows no cross reactivity to human and dog C-Peptide.

Test Principle

This EIA kit for determination of mouse C-Peptide I in plasma and urine samples is

based o n a competitive enzyme immunoassay using combination with highly specific

antibody to mouse C-Peptide I and biotin – avidin affinity system. The 96 wells plate is

coated with goat anti rabbit IgG and C-Peptide I standard or samples, biotinylated mouse

C-Peptide I and rabbit anti mouse C-Peptide I antibody are added to the wells for

competitive immunoreaction. After rinsing out excess mouse C-Peptide I, HRP labeled

streptoavidins are added to bind to the antigen-antibody complex so that HRP labeled

streptoavidin - biotinylated mouse C-Peptide – antibody complexs are formed on the surface

on the wells. Finally, excess HRP labeled streptoavidins are rinsed out and HRP enzyme

activity is determined and the concentration of mouse C-Peptide I is calculated.

V

. Composition

Component Form Quantity Main Ingredient

1. Antibody coated plate MTP

*1 1 plate(96 wells) Goat anti rabbit IgG

2. C-Peptide standard lyophilized 1 vial Synthetic mouse C-Peptide I( 50ng)

3. Labeled antigen lyophilized 1 vial Biotinylated mouse C-Peptide I

4. C-Peptide antibody liquid 1 bottle (6 mL) Rabbit anti mouse C-Peptide I

5. SA-HRP solution liquid 1 bottle (12 mL) HRP labeled streptoavidin

6. Substrate buffer liquid 1 bottle (26 mL) 0.015% Hydrogen Peroxide

7. OPD tablet tablet 2 tablets o-Phenylenediamine hydrochloride

8. Stopping solution liquid 1 bottle (12 mL) 1M-H

2SO4

9. Buffer solution liquid 1 bottle (20 mL) Phosphate buffer

10. Washing solution liquid 1 bottle (50 mL) Concentrated saline

(concentrated)

11. Adhesive foil 3 sheets

MTP

*1 ….. Microtiration plate

W

. Method

Equipment required

1) Photometer for microtitration plate(Plate reader),which can read extinction 2.5 at

492nm

2) Rotator for microtitration plate

3) Washing device for microtitration plate and dispenser for approximately 0.35 mL with

aspiration system

4) Micropipettes, multi-channel pipettes for 8 wells or 12 wells and their tips

5) Test tubes for preparation of standard solution

6) Graduated cylinder(1,000 mL)

7) Distilled water or deionized water

< Preparatory work >

1) Preparation of standard solution:

Reconstitute the C-Peptide standard(lyophilized mouse C-Peptide I 50ng/vial) with 1mL of

buffer solution, which affords 50 ng/mL standard solution. 0.1ml of the reconstituted

standard solution is diluted with 0.2 mL of buffer solution, that yields 16.667 ng/mL

standard solution. 0.1mL of 16.667 ng/mL standard solution is diluted with 0.2 mL of the

buffer solution, that makes 5.556 ng/mL standard solution. Repeat the dilution to make

each standard of 1.852, 0.617 ng/mL. Buffer solution is used as 0 ng/mL.

2) Preparation of labeled antigen:

Reconstitute labeled antigen with 11mL of buffer solution.

3) Preparation of substrate solution:

Resolve OPD tablet with 12 mL of substrate buffer. It should be prepared immediately

before use.

4) Preparation of washing solution:

Dilute 50 mL of washing solution (concentrated) to 1000 mL with distilled or deionized

water.

5) Other reagents are ready for use.

Procedure

1. Warm up the reagents to room temperature (20 - 30°C) before beginning the test at least

for one hour.

2. Fill 25µL of each of standard solutions ( 0, 0.617, 1.852, 5.556, 16.667, 50 ng/mL) or

samples into wells first, then add 100µL of labeled antigen and finally introduce 50µL of

C-Peptide I antibody into the wells .

3. Cover the plate with adhesive foil and incubate it at 4°C for 16 - 18 hours.

4. Incubate it 1 hour at room temperature. During the incubation, the plate should be

rotated with a plate rotator.

5. Take off the adhesive foil, aspirate the solution in the wells and wash the wells three

times with approximately 0.35 mL/well of washing solution.

6. Pipette 100µL of SA-HRP solution into the wells.

7. Cover the plate with adhesive foil and incubate it at room temperature for 1 hour.

During the incubation, the plate should be rotated with a plate rotator.

8. Take off the adhesive foil, aspirate and wash the wells five times with approximately

0.35 mL/well of washing solution.

9. Add 100µL of substrate solution into the wells, cover the plate with adhesive foil and

incubate it for 30 minutes at room temperature.

10. Add 100µL of stopping solution into the wells to stop reaction.

11. Read the optical absorbance of the wells at 492nm.

12. Calculate mean absorbance values of wells containing standards and plot a standard

curve on semilogarithmic graph paper (abscissa: concentration of standard; ordinate:

absorbance values.).

13. Use the standard curve to read C-Peptide concentrations in samples from the

corresponding absorbance values.

X

. Notes

1. Plasma and serum samples must be used as soon as possible after collection. If the

samples are to be tested at a later time, they should be divided into test tubes in small

amount and frozen at or below – 30°C. Avoid repeated freezing and thawing of plasma or

serum samples. EDTA plasma is recommended to use for the determination.

2. C-Peptide standard, labeled antigen, substrate solution should be prepared immediately

before use in assay using clean test tubes or vessels. Diluted washing solution is stable

for 6 months at 2 to 8°C.

3. During storage of washing solution (concentrated) at 2 to 8°C, precipitates may be

observed, however they will be dissolved when diluted.

4. As pipetting operations may affect with the precision of the assay, pipette precisely

standard solutions or samples into each well of plate. And use new tip for each sample to

avoid cross contamination.

5. When sample value exceeds 50 ng/mL, it needs to be diluted with buffer solution within

the assay range.

6. During incubation except 4°C and color reaction, the test plate should be rotated gently

by plate rotator to promote immunoreaction.

7. The plate can be used for separately twice, then reconstituted reagents( standard and

labeled antigen) should be stored at 4°C within 1 week and stored at or less than -30°C

more than 1 week.

8. Read optical absorbance of reaction solution in wells as soon as possible after stopping

the color reaction.

9. Perform all the determination in duplicate.

10. To quantitate accurately, always run a standard curve when testing samples.

11. Protect reagents from strong light (e.g. direct sunlight) during storage and assay.

12. Satisfactory performance of the test is guaranteed only when reagents are used from

combination pack with identical lot number.

Y

. Performance Characteristics

Typical standard curve

Precision and reproducibility

l

Intra-assay CVij : serum 3.1 ~ 4.9

: plasma 5.6 ~ 7.5

: urine 3.4 ~ 4.6

l

Inter-assay CVij : serum 4.7 ~ 8.8

: plasma 4.8 ~ 8.1

: urine 5.3 ~ 11.3

Assay range

0.617 – 50 ng/mL

Analytical recovery

 3 serum, 4 plasma and 4 urine samples

Sample No.          Mouse      C-Peptide I added                   Observed                        Expected                Recovery

                            ng/mL                  ng/mL              ng/mL

Serum 1

                      0.00                   1.97

                             1.85                   3.82                    3.82         100.0

                              5.56                   8.60                  7.53           114.2

                             1 6 . 67               23.03                 18.64          123.6

Serum 2

                     0.00                  1.89

                              1.85                 3.73                  3.74              99.7

                               5.56                7.94                   7.44            106.7

                               1 6 . 6 7           22.22                   18.55         119.8

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

0.1 1 10 100

pg/ml

OD492ABS

 

Sample No. Mouse C-Peptide I added Observed Expected Recovery

ng/mL ng/mL ng/mL

Serum 3

0.00 2.32

1.85 4.06 4.17 97.3

5.56 8.40 7.87 106.7

1 6 . 6 7 22.38 18.98 117.9 

Plasma 1 0.00 1.82

1.85 3.64 3.67 99.2

5.56 7.55 7.37 102.4

1 6 . 6 7 22.95 18.49 124.1

Plasma 2

0.00 1.97

1.85 3.42 3.60 95.0

5.56 7.87 7.31 107.7

1 6 . 6 7 22.30 18.42 121.1

Plasma 3

0.00 1.34

1.85 3.15 3.19 98.6

5.56 7.17 6.89 104.0

1 6 . 6 7 19.73 18.01 109.6

Plasma 4

0.00 1.71

1.85 3.60 3.57 100.9

5.56 7.53 7.27 103.5

1 6 . 6 7 18.45 18.38 100.4

Urine 1

0.00 1.66

1.85 2.47 2.51 98.4

5.56 6.57 6.21 105.8

1 6 . 6 7 19.17 17.32 110.7

Urine 2

0.00 1.25

1.85 2.91 3.10 93.7

5.56 6.83 6.80 100.4

1 6 . 6 7 20.09 17.92 112.1

Urine 3

0.00 2.24

1.85 3.59 4.08 88.0

5.56 7.61 7.79 97.7

1 6 . 6 7 22.34 18.90 118.2

 

Z

. Stability and Storage

< Storage > Store all of the components at 2 to 8°C.

< Shelf life > 6 month from the date of manufacturing

The expiry date is described on the label of kit.

< Package > For 96 tests per 1 kit including standards

[

. References

1. Markussen, J. and Sundby, F. (1972): Eur. J. Biochem., 25: 153.

2. Massey, D. E. and Smyth, D. G. (1975): J. Biol. Chem. 250: 6288.

3. Miyachi, Y. Vaitukais, J. L. Nieschlag, E. and Lipsett, M. B. (1972): J. Clin. Endocr.,34:

23.

4. Smyth, D. G., Markussen, J. and Sundby, F. (1974): Nature(Lond), 248: 151.

5. Tager, H. S., Emdin, S.O., Clark, J. L. and Steiner, D. F. (1973): J. 248: 3476.

6. Tager, H. S. and Steiner, D. F. (1972): J. Biol. Chem., 247, 7936.

7. Yanaihara, N., Sakagami, M., Sakura, N., Iizuka, Y., Nishida, T., Hashimoto, T. and

Yanaihara, C. (1977): In: Deabetes. P116 Ed J.S.Bajaj. Excerpta Media, Amsterdam.

8. Yanaihara, N., Nishida, T., Sakagami, M., and Yanaihara, C. (1977): In: Peptide

Chemistry 1976, p85 Ed T. Nakajima. Protein Research Foundation, Osaka.

9. Yanaihara, N., Yanaihara, C., Sakagami, Sakura, N., Hashimoto, T. and Nishida, T

(1978): , 27, (Suppl 1 ) 149.

10. Yanaihara, C., Ozaki, J., Nishida, T , Iizuka, Y., Sato H., Yanaihara, N., and Kaneko, T..

(1979): Eds. S. Baba, T. Kaneko, N. Yanaihara, p87. Excerpta Medica, Amsterdam.–

Oxford

11. Luo, W. Q., Kanno, T., Winarto, A, Iwanaga, T., Li., J., Futai, Y. Yanaihara, C., and

Yanaihara, N.(1998) : Biomed, Res., 19, 127

10

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